si-RNA-mediated gene silencing technology to inhibit tyrosinase and reduce pigmentation

ABSTRACT

The present invention describes compositions and methods for treating, preventing and improving hyperpigmentation, or other unwanted pigmentation of the skin, or other unwanted skin condition, such as age spots, aged skin, skin discoloration, etc., wherein the compositions include siRNA-gene silencing oligomers specific for tyrosinase. The compositions are used to treat a broad variety of pigmentation conditions, and are preferably applied to the skin, or are delivered by directed means to a site in need thereof.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for skin andpersonal care involving the use of small interfering RNA (siRNA)oligomers for treating, preventing, or ameliorating hyperpigmentation,or other unwanted pigmentation, or other unwanted conditions in humanskin. More particularly, the present invention relates to siRNAoligomers that inhibit the production of tyrosinase, a major enzymeinvolved in melanin production. The invention further relates to methodsof delivery for such compositions so as to allow the siRNA oligomers tomore readily reach the skin layer to improve the aesthetic appearance ofthe skin.

BACKGROUND OF THE INVENTION

Human skin color is determined primarily by the content of the pigmentmelanin in the basal epidermis layer. Melanin is synthesized by theprocess of melanogenesis within melanocytes (pigment-producing cells).Melanin is deposited onto melanosomes, which are transferred tokeratinocytes in the basal epidermal layer. Melanosomes present in thesebasal keratinocytes are the key determinants of skin color. Thekeratinocytes leave the basal layer and undergo differentiation formingthe cornified top layer of the skin. Once the keratinocytes leave thebasal layer, the melanosomes lose their characteristic electron densestructure, and the load of melanin is carried to the surface of the skinby the differentiating keratinocytes.

The skin can become hyperpigmented when too much melanin concentrates atone area or portion of the skin due to the retention time of themelanosomes in the basal layer. Hyperpigmentation can also occur as aresult of overexposure to the sun or other inflammatory stimuli.Hyperpigmentation can take the form of solar lentigines (age spots),ephilides (freckles), melasma, chloasma, and pigmented keratoses.

The prior art discloses ways to treat hyperpigmentation by applicationof skin lightening agents. Representative skin lightening agents includehydroquinone and Vitamin C. Such agents typically lighten the skin byinhibiting the expression of tyrosinase enzymes. Tyrosinase is a majorenzyme involved in the synthesis of melanin in melanosome cells ofhumans and mice. Its activity is linked to, for example, hair and skinpigmentation, and hyperpigmentation, age spots, and uneven skincoloration.

A powerful tool used to study gene function in mammalian cells is theprocess of small interfering RNA (siRNA)-mediated gene silencing. siRNAgene silencing is directly related to the process of RNA interference(RNAi) that is currently being done in Caenorhabditis elegans species ofnematode. The process uses double-stranded RNA that is less than 30 basepairs long, and has a sequence complementary to the messenger RNA (mRNA)targeted. As the siRNA crosses the plasma membrane, the reaction of thecell is to destroy the siRNA and any sequence exactly like it. NativemRNA will also be destroyed by the cell when the siRNA code is the sameas a specific region found on the native mRNA. Thus, the siRNA“silences” expression of a gene product by specifically destroying itsmRNA. Numerous groups describe methods of specifically targeting avariety of endogenously and exogenously expressed genes using siRNA.(Brummelkamp et al. (2002) Science, 296: 550-553; Elbashir et al. (2001)Nature, 411:: 494-498; Paul et. al. (2002) Nature Biotech., 20:505-508;and Hannon, P. (2002) Nature, 418: 244-251). Gene silencing technologyis also being applied in a broad range of therapeutic applications.

Safe, effective and new compositions containing siRNA oligomers totreat, prevent, reduce, inhibit, and/or improve hyperpigmentation, orother unwanted pigmentation in skin and hair, would be advantageous forthe formulation of treatments and products for the skin. As describedherein, novel and beneficial methods and compositions, as well as theirmode of action, for the treatment of hyperpigmentation, or otherunwanted pigmentation and the like, as well as for personal careproducts for the skin, are provided by the present invention.

SUMMARY OF THE INVENTION

In its broadest aspect, the present invention relates to compositionsand methods comprising double-stranded small interfering RNA oligomers(siRNA) to inhibit the production of tyrosinase in a subject. The siRNAinhibit production of the tyrosinase protein by binding to a specificcomplement sequence found in the tyrosinase mRNA. As described above,tyrosinase is a major enzyme found in the pathway of melanin production;therefore, by inhibiting its formation, melanin and pigmentationdecreases. Thus, the compositions and methods of the invention can beused to treat, including prevent, reduce, ameliorate and/or eliminate,hyperpigmentation and/or unwanted pigmentation in human skin, therebyimproving the aesthetic appearance of skin.

In a specific embodiment, the present invention relates to an siRNAconstruct that is 21 oligonucleotides in length and has the followingsequence:

5′-UAGGACCUGCCAGUGCUCUtt-3′ (SEQ ID NO: 1) 3′-ttAUCCUGGACGGUCACGAGA-5′.(SEQ ID NO: 2) 5′-UAGGACCUGCCAGUGCUCUtt-3′ (SEQ ID NO: 1)3′-ttAUCCUGGACGGUCACGAGA-5′ (SEQ ID NO: 2) 5′-UCCUGGAAACCAUGACAAAtt-3′(SEQ ID NO: 3) 3′-ttAGGACCUUUGGUACUGUUU-5′ (SEQ ID NO: 4)5′-CACACCUGUCUUUGUCUUAtt-3′ (SEQ ID NO: 5) 3′-ttGUGUGGACAGAAACAGAAC-5′(SEQ ID NO: 6)

There are multiple sequences possible for the siRNA oligomers of theinvention, but they preferably begin with two adenosines and are a totalof 21 base pairs long. The first two siRNAs shown above are homologousto sequences found in both human and mouse forms of tyrosinase. Thethird siRNA is just one example of many from the human sequence oftyrosinase.

The siRNA for tyrosinase when applied to the skin will prevent, reduce,ameliorate and/or eliminate hyperpigmentation, or other unwantedpigmentation, or other unwanted conditions in human skin and hair,thereby lightening skin tone, bleaching or lightening hair, decreasinghyperpigmented states such as age spots, and improving skindiscoloration.

It is to be understood that, as used herein, the terms treating andtreatment include and encompass preventing, reducing, ameliorating,improving, alleviating, and/or eliminating hyperpigmentation, or otherunwanted pigmentation of the skin, and the like. The presentcompositions and methods are also suitable for use in treating, asdefined above, hyperpigmentation, or other unwanted pigmentation, of theskin and hair in numerous areas of the body, including, withoutlimitation, the face, forehead, neck, arms, hands, legs, knees, feet,chest, back, groin, buttocks, and the like.

It is another aspect of the present invention to provide compositions,formulations and methods containing siRNA oligomers in the treatment ofpigmentation, hyperpigmentation, unwanted hair pigmentation, age spots,and/or uneven skin tone, etc.. These oligomers exert their effectivenessaccording to this invention by preferably crossing the plasma membraneof skin cells, wherein the siRNA and any sequence exactly like it (inthis case, tryosinase mRNA) will be destroyed and degraded at the siteof application, e.g., skin of the face, neck, arms, feet, or hands,particularly in the lower epidermis/upper dermis, and the layer of theskin where hair follicles/melanocytes interact. More specifically, thesiRNA oligomers block or inhibit the native human tyrosinase mRNA,which, in turn, results in an inhibition of tyrosinase enzyme productionand a reduction of pigmentation because the tyrosinase enzyme will notbe present for melanin synthesis.

In addition, because it is understood that melanin synthesis is relatedto pigmentation, hyperpigmentation, hair pigmentation, age spots, etc.,the inhibition, and/or the control or modulation of the proteinsinvolved in melanin production by the newly-determined action of thesiRNA oligomers as disclosed herein can serve a pivotal function in thetreatment, prevention, reduction, amelioration, or elimination ofunwanted pigmentation, and the like.

In accordance with this invention, the siRNA oligomers comprisecompositions which include, without limitation, topically appliedsunscreens, anti-oxidants, anti-inflammatories, cosmetics, includingmakeups, skin care actives, e.g, for fine lines and/or wrinkles and thelike. Also in accordance with this invention, ingredients, components,or compounds that are formulated in such compositions in a variety ofproduct forms, e.g., transdermals, such as patches, and the like, areencompassed, particularly for topical administration.

Another aspect of the present invention provides the compositionscomprising the siRNA oligomers preferably for topical administrationwithout inducing significant irritation. Further, such compositions arepreferably delivered by, but not limited to, the use of targeteddelivery systems, for example, liposomes, microspheres, transdermalpatches, lipid or protein delivery systems, and the like, so that theactives can more readily reach and affect the melanin producing cells ofthe skin in the area of application, e.g., face, hands, or neck, or thedermal layer of the skin, where hair follicles are located. Compositionscomprising siRNA oligomers, including liposome formulations, can beadministered topically and through skin pores to deliver the siRNAoligomers to the sites requiring treatment.

In another of its aspects, the present invention provides compositionscontaining one or more siRNA oligomers and methods thereof which canimprove the aesthetic appearance of the skin by treating, includingpreventing, eliminating, ameliorating and/or reducing at least one ofthe following: pigmentation, hyperpigmentation, age spots, dark circlesunder the eyes, hair pigmentation in an area where hair is not wanted,and mottled pigment. The improvement preferably results followingtopical application of a product or formulation containing an effectiveamount of one or more of the siRNA oligomers as described herein.

Another aspect of this invention provides a method of reducing,preventing, treating, or ameliorating one or more skin conditions due todermatological aging or photoexposure of skin, comprising: topicallyapplying a composition comprising one or more siRNA oligomers in anamount effective to reduce or block native tyrosinase mRNA, wherein thereduction or block of native tryrosinase mRNA concomitantly reduces orblocks melanin production, thereby treating, preventing, reducing,ameliorating, or eliminating hyperpigmentation, or other unwantedpigmentation, or other unwanted skin condition.

Further aspects, features and advantages of the present invention willbe better appreciated upon a reading of the detailed description of theinvention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 presents the results of a Northern Blot experiment run to measurethe amount of tyrosinase mRNA in B16 mouse melanoma cell line after 48hours of treatment with a tyrosinase siRNA.

DESCRIPTION OF THE INVENTION

The present invention provides novel compositions and methods comprisingsiRNA oligomers herein found to be effective to treat, includingprevent, reduce, ameliorate, inhibit, alleviate, and/or eliminatehyperpigmentation, or other unwanted pigmentation, due to dermatologicalaging of skin, due to chronological and/or hormonal aging, or due tophotoexposure (e.g., to the sun, or ultraviolet radiation) of skin,and/or to improve the aesthetic appearance of skin.

More particularly, the present invention relates to the use of siRNAoligomers that are less than 30 oligonucleotides in length, morepreferred between 15 and 30, more preferred between 19 and 25, andmostly preferred between 19 and 22 oligonucleotides in length, and havea sequence complementary to native human tyrosinase mRNA. In a specificembodiment, the present invention relates to an siRNA oligomer that is21 oligonucleotides in length and has the following sequence:

5′-UAGGACCUGCCAGUGCUCUtt-3′ (SEQ ID NO: 1) 3′-ttAUCCUGGACGGUCACGAGA-5′.(SEQ ID NO: 2) 5′-UAGGACCUGCCAGUGCUCUtt-3′ (SEQ ID NO: 1)3′-ttAUCCUGGACGGUCACGAGA-5′ (SEQ ID NO: 2) 5′-UCCUGGAAACCAUGACAAAtt-3′(SEQ ID NO: 3) 3′-ttAGGACCUUUGGUACUGUUU-5′ (SEQ ID NO: 4)5′-CACACCUGUCUUUGUCUUAtt-3′ (SEQ ID NO: 5) 3′-ttGUGUGGACAGAAACAGAAC-5′(SEQ ID NO: 6)

According to the present invention, yet without wishing to be bound bytheory, the siRNA oligomers exert their effects by their ability toinhibit, block, reduce, or prevent the translation of native tyrosinaseenzyme mRNA. The ability of the siRNA oligomers to inhibit or silencetyrosinase enzyme production results in an inhibition or modulation ofmelanin production so as to decrease pigmentation in skin or hair,thereby, lightening hyperpigmentation, or other unwanted pigmentation,and the like.

Thus, in one of its embodiments, the present invention encompassescompositions, formulations and methods containing siRNA, hereindetermined to be useful in the treatment of hyperpigmentation, or otherunwanted pigmentation of skin or hair. Skin or hair in a variety ofareas of the body is amenable for treatment and/or receipt of thecompositions of the present invention, including the face, forehead,neck, arms, legs, hands, feet, torso (chest), back, and the like.

The siRNA molecules of the present invention can be obtained using anumber of techniques known to those of skill in the art. For example,the siRNA can be chemically synthesized or recombinantly produced usingmethods known in the art. For example, short sense and antisense RNAoligomers can be synthesized and annealed to form double-stranded RNAstructures with 2-nucleotide overhangs at each end (Caplen, et al.(2001) Proc Natl Acad Sci USA, 98:9742-9747; Elbashir, et al. (2001)EMBO J, 20:6877-88). These double-stranded siRNA structures can then bedirectly applied to the skin and enter cells, either by passive uptakeor a delivery system of choice, such as described below. Some modes ofsynthesis include chemically synthesizing strands and annealing at alater date, in vitro transcription through the use of polymerase chainreaction (PCR) and Dnase digestion, or through the insertion of siRNAsequences in to plasmids (Brummelkamp et al. (2002) Science,296:550-553; Jarvis et al., Ambion, Inc., poster published on the worldwide web at ambion.com/techlib/posters/RNAi_(—)0302.html)

The siRNA molecules can be purified using a number of techniques knownto those of skill in the art. For example, gel electrophoresis can beused to purify siRNAs. Alternatively, non-denaturing methods, such asnon-denaturing column chromatography, can be used to purify the siRNA.In addition, chromatography (e.g., size exclusion chromatography),glycerol gradient centrifugation, and affinity purification withantibody can be used to purify siRNAs.

In a preferred embodiment, the siRNA oligomers are stabilized againstdegradation because of their double stranded nature and the introductionof Dnase/Rnase inhibitors. For example, the siRNA can be stabilized byincluding thymidine or uridine nucleotide 3′ overhangs.

The siRNA contained in the compositions of the present invention can bechemically synthesized at industrial scale in large amounts. Methodsavailable would be through chemical synthesis, or through the use of abiological agent.

The present invention encompasses compositions comprising one or moresiRNA oligomers, preferably in a pharmaceutically-acceptable. cosmetic,or dermatological formulation which is suitable for contact with livinganimal tissue, including human tissue, and for topical administration,with virtually no adverse physiological effect, e.g., irritation, to theuser. Thus, the inventive compositions are especially suitable forsensitive skin.

Compositions embraced by this invention can be provided in anycosmetically and/or dermatologically suitable form, for example, anemulsion, a cream, a mousse, a gel, a foam, a lotion, a mask, anointment, a pomade, a solution, a serum, a spray, a stick, a patch, or atowelette. In addition, the compositions contemplated by this inventioncan include one or more compatible cosmetically acceptable additionalingredients, such as colorants, fragrances, emollients, humectants,preservatives, vitamins, thickeners, anesthetics, anti-acne agents,anti-allergenics, antifungals, antimicrobials, anti-inflammatory agents,anti-irritants, antineoplastics, antioxidants, antiseptics, antivirals,chelating agents, depigmenting agents, emulsifiers, exfollients, filmformers, hypopigmenting agents, immune system boosting agents, immunesystem suppressing agents, insect repellents, lubricants, moisturizers,pharmaceutical agents, photostablizing agents, preservatives, retinoids,skin protectants, skin penetration enhancers, staining agents,sunscreens, stabilizers, surfactants, viscosity and/or rheologymodifiers, and the like, as well as other botanicals such as aloe,chamomile, and the like, and as further described below. Thecompositions are applied topically for an effective period of time,preferably at least once or twice daily, for at least one week. Thedaily application can be for periods of up to two weeks, four weeks, ormore.

The compositions of this invention can also be formulated into liposomeswhich can comprise other additives or substances, and/or which can bemodified to more specifically reach or remain at a site followingadministration. Although the current system in use is a lipid basedsystem, this invention also contemplates other delivery systems, forexample, protein delivery systems or microspheres if the cell membraneis to be crossed.

Dermatologically acceptable compositions suitable for use in the presentinvention include compositions in which the active constituents,ingredients, or materials are contained in an amount effective toachieve the intended purpose. By way of example, in the presentcompositions, a siRNA oligomer is present in an amount of from about0.0001 wt % to about 10 wt %, based on the total weight of thecomposition. More preferably, the present compositions include one ormore siRNA oligomers in an amount from about 0.0005 wt % to about 5 wt%. Most preferably, the present compositions include one or more siRNAoligomers in an amount from about 0.001 wt % to about 1 wt % of thetotal composition.

The determination of an effective dose or amount is well within thecapability of those skilled in the art. A therapeutically effective doserefers to that amount of siRNA oligomer identified in accordance withthe present invention, which, for instance, treats, prevents,ameliorates, reduces, or eliminates hyperpigmentation, or other unwantedpigmentation, and the like. The practitioner, who will consider thefactors related to the individual requiring treatment, will determinethe exact dosage. Dosage and administration are adjusted to providesufficient levels of the siRNA oligomer to bring about the desiredeffect. Factors which are typically considered include the severity ofthe degree of penetration into the skin, the individual's particularneed, time and frequency of administration, drug combination(s),reaction sensitivities, and tolerance/response to treatment. Variationsin dosage levels can be adjusted using standard empirical routines foroptimization, as is well understood in the art. The range is usuallybetween 1 nm to 100 μm (Elbashir et al. (2001) Nature, 411:494-498).Those skilled in the art will employ different formulations dependingupon the nature, e.g., structure, composition, of the siRNA.

Embraced by the present invention are transdermal modes of delivery,such as patches and the like, with or without a suitable skinpenetration enhancer. The methods and compositions embodied by theinvention provide a means by which the siRNA oligomers can beeffectively administered in a transdermal system. Frequently, compoundshaving poor topical absorption, or which are required at high dosagelevels, are delivered transdermally. Accordingly, a transdermal means ofdelivering a composition or formulation (often with a skin penetrationenhancer composition) to the skin is that of the transdermal patch or asimilar device as known and described in the art. Examples of suchdevices are disclosed in U.S. Pat. Nos. 5,146,846, 5,223,262, 4,820,724,4,379,454 and 4,956,171; such descriptions are not meant to be limiting.The transdermal mode of storing and delivering the compositions onto theskin and forming the active composition is convenient and well suitedfor the purposes of an embodiment of the present invention. Preferably,when a topical patch is used, the patch is applied to the desired areafor an extended period of time. Preferably, the extended period of timeis greater than one hour; more preferably, the extended period of timeis overnight, e.g., when the user is sleeping.

A particular embodiment of the present invention is directed to thedelivery of the described compositions by the use of targeted deliverysystems, for example, liposomes, microspheres (see, e.g., U.S. Pat. No.5,770,222 to Unger et al.; Rozema, D. and Lewis, D. (2003) Targets, 2:253-260), and the like, so that the siRNA can more readily reach andaffect the skin cells of the area of application, e.g., face or neck, orthe lower epidermis/upper dermis layer of the skin, where hair folliclesand melanocytes are located together. Compositions comprising siRNAoligomers, including liposome formulations, can also be administered bydirect injection subcutaneously or intradermally to more preciselydeposit the active agents.

Liposomes and delivery systems and vehicles involving liposomes arewell-known in the art. In brief, liposomes are unilamellar ormultilamellar lipid vesicles which entrap a significant fraction ofaqueous solution. The vesicular microreservoirs of liposomes can containa variety of water-soluble materials, which are suspended within theemulsion (e.g., reviewed in G. Gregorius (Ed.), 1991, LiposomeTechnology, Vols. I, II, III, CRC Press, Boca Raton, Fla.; Davis S. S.and Walker I. M., 1987, Methods in Enzymology, 149:51-64; Mayhew E. etal., 1987, Methods in Enzymology, 149:64-77; and Shafer-Korting M. etal., 1989, J. Am. Acad. Dermatol., 21:1271-1275). The preparation ofliposomes and the variety of uses of liposomes in biological systemshave been described (e.g., in U.S. Pat. No. 4,708,861 to M. C. Popescuet al., U.S. Pat. No. 4,224,179 to M. Schneider and U.S. Pat. No.4,235,871 to D. P. Papahadjopoulos et al.). Accordingly, such liposomescan be formulated into any of the dermatological or cosmeticcompositions as described herein.

The siRNA oligomers of the present invention can also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example,biodegradable polymers or receptor targeted molecules for topical andother formulations.

In addition to the siRNA as active agents, as described herein, thephysiologically acceptable compositions can contain suitablephysiologically acceptable carriers, diluents, or excipients comprisingauxiliaries which facilitate processing of the siRNA oligomers intopreparations which can be used cosmetically and/or pharmaceutically.Further details on techniques for formulation and administration areprovided in the latest edition of Remington's Pharmaceutical Sciences(Mack Publishing Co.; Easton, Pa.). Compositions containing the siRNAoligomers of the present invention can be manufactured in a manner thatis known in the art, e.g., by means of conventional mixing, dissolving,granulating, dragee-making, levigating, emulsifying, encapsulating,entrapping, or lyophilizing processes.

A preferred embodiment of the topical compositions of the presentinvention may also include at least one of the following: a surfacesmoother, a skin plumper, an optical diffuser, a sunscreen, anexfoliation promoter, or an antioxidant.

A surface smoother provides the functional benefits of enhancing skinsmoothness and reducing the appearance of fine lines and coarsewrinkles. Examples include, without limitation, isopropyl myristate,petrolatum, isopropyl lanolate, silicones (e.g., methicone,dimethicone), or any mixtures thereof. The surface smoother ispreferably present from about 0.1 wt % to about 50 wt % of the totalweight of the composition. A skin plumper serves as a collagen enhancerto the skin. An example of a suitable and preferred skin plumper ispalmitoyl oligopeptide. Other nonlimiting examples of skin plumpersinclude collagen and/or glycosaminoglycan (GAG) enhancing agents. Theskin plumper is preferably present from about 0.1 wt % to about 20 wt %of the total weight of the composition.

An optical diffuser is a particle that changes the surface optometriesof skin, resulting in a visual blurring and softening of, for example,lines and wrinkles. Examples of optical diffusers that can be used inthe present invention include, but are not limited to, boron nitride,mica, nylon, polymethylmethacrylate (PMMA), polyurethane powder,sericite, silica, silicone powder, talc, Teflon, titanium dioxide, zincoxide, or any mixtures thereof. The optical diffuser is preferablypresent from about 0.01 wt % to about 20 wt % of the total weight of thecomposition.

In another embodiment, the present invention embraces a sunscreen thatprotects skin from damaging ultraviolet rays. Illustratively, thesunscreen provides both UVA and UVB protection by using either a singlesunscreen or a combination of sunscreens. Among the sunscreens that canbe employed in the present compositions are avobenzone, cinnamic acidderivatives (such as octylmethoxy cinnamate), octyl salicylate,oxybenzone, titanium dioxide, zinc oxide, or any mixture or combinationthereof. Preferably, the sunscreen is present from about 1 wt % to about30 wt % of the total weight of the composition. In particular, theaddition of a sunscreen is preferred to prevent/reduce thephotodegradation of the composition and/or ingredients therein while inthe package and/or on the skin after application.

It will be appreciated that the compositions of the present inventioncontaining sunscreen bring about additional improvements to theaesthetic appearance of skin, including at least one of the following:minimizes sunburning, minimizes tanning, reduces redness, and reducesfuture wrinkle development. It will be appreciated that when the topicalcomposition is intended to be applied prior to retiring for the evening,the addition of a sunscreen agent may not be required.

The present compositions may also have one or more skin active agents,such as exfoliation promoters. Suitable examples that can be used in thepresent compositions include keratolytic agents, i.e., an active agenthaving desquamating, exfoliant, or scrubbing properties, or an activeagent which can soften the horny layer of the skin; alpha (α) and/orbeta (β) hydroxy acids; benzoyl peroxide; keto acids, such as pyruvicacid, 2-oxopropanoic acid, 2-oxobutanoic acid, and 2-oxopentanoic acid;oxa acids, as disclosed in U.S. Pat. Nos. 5,847,003, 6,069,169,5,932,229 and 5,834,513, the disclosures of which are incorporatedherein by reference; urea; retinoids, or any mixtures thereof. Theseanti-wrinkle or anti-fine line active agents can be formulated, forexample, in amounts of from about 0.0001% to 5% by weight relative tothe total weight of the composition.

More specifically, examples of hydroxy acids include, but are notlimited to, glycolic acid, lactic acid, malic acid, tartaric acid,citric acid, 2-hydroxyalkanoic acid, mandelic acid, salicylic acid andalkyl derivatives thereof, including 5-n-octanoylsalicylic acid,5-n-dodecanoylsalicylic acid, 5-n-decanoylsalicylic acid,5-n-octylsalicylic acid, 5-n-heptyloxysalicylic acid,4-n-heptyloxysalicylic acid and 2-hydroxy-3-methylbenzoic acid or alkoxyderivatives thereof, such as 2-hydroxy-3-methyoxybenzoic acid. Exemplaryretinoids include, without limitation, retinoic acid (e.g., all-trans or13-cis) and derivatives thereof, retinol (Vitamin A) and esters thereof,such as retinol palmitate, retinol acetate and retinol propionate, andsalts thereof. Preferred skin active agents are3,6,9-trioxaundecanedioic acid, glycolic acid, lactic acid, or anymixtures thereof.

When the present invention includes a skin active agent, the compositiontypically includes about 0.5 wt % to 30 wt %, preferably about 1 wt % toabout 15 wt %, more preferably about 2 wt % to about 10 wt %, and mostpreferably about 4 wt %, of the skin active agent based on the totalweight of the composition.

An antioxidant functions, among other things, to scavenge free radicalsfrom skin to protect the skin from environmental agressors. Examples ofantioxidants that can be used in the present compositions includecompounds having phenolic hydroxy functions, such as ascorbic acid andits derivatives/esters; beta-carotene; catechins; curcumin; ferulic acidderivatives (e.g., ethyl ferulate or sodium ferulate); gallic acidderivatives (e.g., propyl gallate); lycopene; reductic acid; rosmarinicacid; tannic acid; tetrahydrocurcumin; tocopherol and its derivatives;uric acid; or any mixtures thereof. Compositions of the presentinvention can include an antioxidant preferably from about 0.001 wt % toabout 10 wt %, and more preferably from about 0.001 wt % to about 5 wt%, of the total weight of the composition.

The compositions of this invention can also include one or moreingredients, additives, or adjuvants as described in detail above. Theamounts of these various ingredients are those that are conventionallyused in the cosmetic or pharmaceutical fields, for example, they canconstitute from about 0.01% to 20% of the total weight of thecomposition. In addition, these ingredients can be introduced into thefatty phase, into the liquid phase, and/or into lipid vesicles,depending on their nature.

The component(s) of the present invention are preferably contained in acosmetically acceptable medium (i.e., vehicle, diluent or carrier). Inan embodiment embracing topical application, the compositions of thisinvention comprise a medium that is compatible with human skin. Thecompositions can be formulated as aqueous, alcohol, oraqueous/alcohol-based solutions, ointments, lotions, gels, water-in-oil,oil-in-water, or water-oil-water triple emulsions having the appearanceof a cream or gel, microemulsion, or aerosol. In addition, thecompositions can be in the form of vesicular dispersions containingionic and/or nonionic lipids, as described above.

In one embodiment, the present invention relates to the administrationof an effective amount of at least one siRNA oligomer or compositioncomprised thereof to inhibit, block, or silence tyrosinase enzymeproduction in skin and to reduce hyperpigmentation, or other unwantedpigmentation, or other skin condition.

In another embodiment, the present invention encompasses a method oftreating hyperpigmentation, or other unwanted pigmentation, or otherdermatological effects of aging or photoexposure of skin, comprisingapplying to skin a composition containing a siRNA specific fortyrosinase in a cosmetically and/or dermatologically acceptable medium,and in an amount effective to treat, reduce, prevent and/or amelioratehyperpigmentation, or other unwanted pigmentation, or other skincondition. The application of the siRNA-containing composition ispreferably topical. In addition, the composition is preferably appliedvia a directed mode of delivery, for example, by topical application ofan aqueous composition or transdermal patch.

Another embodiment of the present invention relates to a method ofimproving the aesthetic appearance of skin and comprises applying to theskin, or introducing via a directed mode of delivery, a compositionincluding one or more siRNA oligomer in an amount effective to improvethe aesthetic appearance of the skin. According to this embodiment, theimprovement in aesthetic appearance includes, but is not limited to, thetreatment of at least one condition, such as pigmentation,hyperpigmentation such as age spots, unwanted hair pigmentation, darkcircles under the eyes, and uneven skin coloration.

EXAMPLE

The following example illustrates a specific aspect of the invention.The example should not be construed as limiting the invention, as theexample merely provides specific understanding and practice of theinvention and its various aspects.

FIG. 1 presents the results of a Northern Dot Blot experiment run tomeasure the amount of tyrosinase mRNA seen in B16 mouse melanoma cellline after a 48 hour treatment with a tyrosinase siRNA.

The contents of all patents, patent applications, published articles,abstracts, books, reference manuals and abstracts, as cited herein arehereby incorporated by reference in their entireties to more fullydescribe the state of the art to which the invention pertains.

As various changes can be made in the above-described subject matterwithout departing from the scope and spirit of the present invention, itis intended that all subject matter contained in the above description,or defined in the appended claims, be interpreted as descriptive andillustrative of the present invention. Many modifications and variationsof the present invention are possible in light of the above teachings.

1. A method of inhibiting the production of melanin in human skincomprising topically applying to the skin a composition comprising asiRNA consisting of: 5′-UAGGACCUGCCAGUGCUCUtt-3′ (SEQ ID NO: 1) and3-′ttAUCCUGGACGGUCACGAGA-5′ (SEQ ID NO: 2); in an amount effective toinhibit the production of melanin.
 2. The method according to claim 1,wherein the composition is applied to the skin suffering from ahyperpigmenred condition selected from the group consisting of agespots, freckles, skin discoloration, and combinations thereof.
 3. Themethod according to claim 1, wherein the skin is sensitive skin.
 4. Themethod according to claim 1, wherein the composition is appliedtopically at least once daily for at least one week.
 5. The methodaccording to claim 1, wherein the siRNA is present in an amount of fromabout 0.0001 wt % to about 10 wt % of the total weight of thecomposition.
 6. The method according to claim 1, wherein the siRNA ispresent in an amount of from about 0.0005 wt % to about 5 wt % of thetotal weight of the composition.
 7. The method according to claim 1,wherein the siRNA is present in an amount of from about 0.001 wt % toabout 1 wt % of the total weight of the composition.
 8. The methodaccording to claim 1, wherein the composition comprises a cosmeticallyor dermatologically acceptable vehicle.
 9. The method according to claim1, wherein the composition is administered in a liposome deliveryvehicle or a transdermal patch.
 10. The method according to claim 9,wherein the composition is administered in a liposome delivery vehicle.11. The method according to claim 1, wherein the composition isadministered in a biodegradable microsphere.
 12. The method according toclaim 1, wherein the composition further comprises a sunscreen.
 13. Themethod according to claim 12, wherein the sunscreen is selected from thegroup consisting of avobenzone, cinnamic acid derivatives, octylsalicylate, oxybenzone, titanium oxide, zinc oxide and combinationsthereof.
 14. The method according to claim 13, wherein the cinnamic acidderivative is octylmethoxycinnamate.
 15. The method according to claim1, wherein the composition further includes an ingredient selected fromthe group consisting of an alpha hydroxy acid, a beta hydroxy acid, aketo acid, an oxa acid and an oxa diacid.